Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Abstract

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Figure 1

Strategy for genotyping of embryos. Wild-type- (3820 and 3846) or mutant- (RTSC18 and MTA2) specific primers were used to amplify embryonic DNA by PCR. Presence of the normal and mutant alleles was demonstrated by visualization of the corresponding PCR fragments with ethidium bromide.

Figure 2

Wild-type and Tsc2^Ek/Ek mutant embryos. (A) Gross appearance of wild-type (left) and mutant (right) E10.5 embryos. Mutant embryo is smaller and has an abnormal head shape. (B) Head of E10.5 mutant embryo exhibiting dysraphia with protruding folds of neuroepithelium indicated by arrow. (C) Sagittal section of the embryo in B; narrow arrow indicates prominent folding of forebrain (F) and wide arrow indicates opening of midbrain (M) to the amniotic cavity. Hindbrain (H) and choroid plexus were morphologically normal.

Figure 3

In situ hybridization. Sagittal section of E10.5 wild-type embryo showing localization of Tsc2 expression by hybridization of antisense (A) and sense (B) Tsc2–1794 riboprobes to the developing neuroepithelium. F, forebrain; M, midbrain; H, hindbrain. Arrows indicate the neuroepithelium, which was intensely labeled by using antisense Tsc2 riboprobe.

Figure 4

Neuroepithelial abnormalities in mutant embryos. (A) Forebrain of an E10.5 Tsc2^Ek/Ek mutant embryo. (B) Forebrain of wild-type embryo at E10.5, which is more rounded and inflated than the forebrain of mutant embryo shown in A. (C) Head of Tsc2^Ek/Ek embryo at E11.5 with papillary overgrowth of midbrain neuroepithelium, indicated by arrows. Forebrain is misshapen and disorganized. (D) Head of wild-type embryo at E11.5. (A and C) Arrows indicate areas of thickening and folding of neuroepithelium in mutant embryos. F, forebrain.